Design.--Clinical, histologic, and immunophenotypic features of 5
OEMCas and 5 ApEMCas were analyzed. Ultrastructural examination was also
performed on 3 OEMCa and 1 ApEMCa tumors.

Results.--The mean age for OEMCa (74.4 years; range, 58-82 years)
was slightly higher than for ApEMCa (61.6; range, 46-79 years). All
tumors arose in the parotid glands and demonstrated a multinodular
pattern of growth with an average size of 3.3 cm (range, 2.3-6.5 cm).
Available follow-up (n = 6; 3 OEMCas, 3 ApEMCas) shows a favorable
course (no evidence of disease; mean, 17.4 months). Both were
morphologically similar, but only OEMCa had sebaceous elements.
Phosphotungstic acid hematoxylin staining, antimitochondrial antibody
immunohistochemistry, and ultrastructural examination confirm the
abundance of mitochondria in OEMCa but not in ApEMCa. The ductal
component in ApEMCa was distinguished from that of OEMCa by apical
snouts, intracytoplasmic vacuoles, nuclear pleomorphism, prominent
nucleoli, and androgen receptor immunoreactivity.

Conclusions.--Oncocytic-sebaceous epithelial-myoepithelial
carcinoma and ApEMCa should be considered in the differential diagnosis
of oncocytic/oncocytoid salivary gland tumors. Oncocytic-sebaceous
epithelial-myoepithelial carcinoma morphology may reflect a senescent
phenotype, similar to other oncocytic lesions. The ductal component of
ApEMCa shares some similarities with salivary duct carcinoma and
supports the notion that epithelial-myoepithelial carcinoma can serve as
the progenitor tumor for hybrid tumors.

Epithelial-myoepithelial carcinoma (EMCa) is an uncommon, biphasic
salivary gland malignancy composed of ductal epithelial cells and
myoepithelial cells with a broad morphologic spectrum. In the classic
definition of EMCa, the myoepithelial component consists of polygonal
cells with clear cytoplasm, whereas the ductal component is composed of
small lumina lined by cuboidal, mildly eosinophilic cells reminiscent of
intercalated ducts. (1-3) However in our previously reported series of
61 cases, (4) we had observed a much broader morphologic spectrum with
respect to cytoplasmic characteristics, relative cell type proportion,
and pattern of arrangement. As a result, several new and rediscovered
histologic variants emerged.

Among these variants was the oncocytic or oncocytic-sebaceous EMCa
(OEMCa), which was initially described by Savera and Salama (5) in 2005.
This variant, defined by prominent oncocytic change in the ductal and/or
myoepithelial component and sebaceous elements, populated 8% of all our
EMCa. (4) The cells in this variant are true oncocytes because the
prominent granular, eosinophilic cytoplasm is constituted by abundant
mitochondria. Subsequent to that publication, we encountered several
EMCa that were, at first glance, very similar to OEMCa in terms of the
abundant granular, eosinophilic cytoplasm. However, unlike the oncocytes
in OEMCa, these cells could only be considered "oncocytoid"
because they did not quite show the same degree of granularity or
abundant mitochondria by histochemical or immunohistochemical stains.
Additionally, these cells showed periapical snouts, vacuolated
cytoplasm, and nuclei with prominent central nucleoli. These cells were
also positive for androgen receptor (AR), which is typically expressed
in salivary duct carcinoma. We herein describe 5 cases of a new EMCa
variant, which we dub apocrine EMCa (ApEMCa), and compare these tumors
with an equal number of OEMCa.

MATERIALS AND METHODS

Case Selection

This study was approved by the University of Pittsburgh Medical
Center Institutional Review Board (0601084). Five cases (4 consults and
1 in-house case) of OEMCa were retrieved from the Department of
Pathology at University of Pittsburgh Medical Center (2004--2008). Four
of these cases were extracted from our prior series of EMCa, (4) and
detailed clinicopathologic parameters and extended follow-up are
presented here. The major inclusion criterion was oncocytic cytoplasm in
at least 50% of tumor (epithelial and/or myoepithelial cell components)
on hematoxylineosin examination. Five cases of ApEMCa were also
retrieved (4 consults and 1 in-house case). Inclusion criteria were
ductal luminal cells with apocrine morphology (abundant
granular/vacuolated oncocytoid cytoplasm with periapical snouts) and
immunoreactivity for AR. Clinicopathologic parameters were obtained.

For OEMCa, the percentages of oncocytic and sebaceous components
were assessed, whereas for ApEMCa, the percentage of apocrine components
was assessed. Additional histopathologic features were evaluated as
described previously, (4) including architectural growth patterns,
presence of luminal microcalcifications, tumor necrosis, perineural
invasion, and angiolymphatic invasion. Cytologic features of the
epithelial and myoepithelial cells were evaluated, including the degree
of nuclear atypia (mild, moderate, and severe), mitotic rate, and
percentages of epithelial and myoepithelial components.

Ultrastructural Examination

Electron microscopy was performed on formalin-fixed
paraffin-embedded material from 4 cases (3 OEMCa and 1 ApEMCa). A 3-mm3
portion of tissue was mechanically dissected from the paraffin block,
minced into smaller portions, and then post-fixed in 2% osmium
tetroxide. In each case, thick sections were cut and stained with
toluidine blue to select suitable areas for ultrastructural examination
under a Philips 410 transmission electron microscope (FEI Company,
Hillsboro, Oregon).

Histochemistry and Immunohistochemistry

Single-label immunohistochemical stains were performed on all cases
with adequate material. Table 1 summarizes the antibodies used. All
stains were performed on a Ventana Benchmark autostainer (Ventana
Medical Systems, Tucson, Arizona), except the antimitochondrial
antibody, which was performed by hand. Staining was visualized using the
Ventana iVIEW 2'-diaminobenzidine detection kit (Ventana Medical
Systems) as the substrate chromogen (brown) for all antibodies, except
S100 and antimitochondrial antibody. The S100 was visualized using the
Ventana alkaline phosphatase red detection kit (Ventana Medical Systems)
as the substrate chromogen (red). For the antimitochondrial antibody,
traditional 2'-diaminobenzidine (Dako, Carpinteria, California) was
used.

Double immunohistochemical staining for p63 and smooth muscle actin
was also performed. For visualization of smooth muscle actin, nickel
ammonium sulfate was added to the 2'-diaminobenzidine substrate
chromogen to impart a black color. For p63 visualization, the
proprietary Vector NovaRED chromogen (Vector Laboratories, Burlingame,
California) was used to impart a dark red color.

To improve visual clarity of the double stains, the Nuance (CRI,
Inc, Woburn, Massachusetts) multispectral imaging system was used as
previously described.6 Briefly, a liquid-crystal tunable filter
optically coupled to a charge-coupled device camera was used for
acquisition of spectra, after acquisition of a reference
''image cube'' from the white background, and
creation of a spectral library for hematoxylin and each chromogen. The
un mixed spectra were reassigned to construct a
"pseudofluorescent" composite image with a black background,
using the following scheme: green, nickel-2'-diaminobenzidine; red,
Vector NovaRED; and light blue, hematoxylin.

RESULTS

Gross and clinical features are summarized in Table 2. The mean age
at presentation was 68 years (range, 46-82 years). The mean age for
OEMCa (74.4 years; range, 58-82 years) was slightly higher than for
ApEMCa (61.6 years; range, 46-79 years). The female to male ratio was
even overall (OEMCa, 3:2; ApEMCa, 2:3). All tumors arose in the parotid
glands with an average size of 3.3 cm (range, 2.3-6.5 cm).
Oncocytic-sebaceous epithelial-myoepithelial carcinomas were slightly
larger on presentation (mean, 3.8 cm; range, 2.5-6.5 cm) than ApEMCa
(mean, 2.9 cm; range, 2.3-3.4 cm). All patients underwent surgical
resection in the form of a parotidectomy. One patient with OEMCa
received radiation therapy. Follow-up was available for 6 patients (3
OEMCa, 3 ApEMCa). These patients had no evidence of disease with a mean
follow-up of 17.4 months (range, 4.3-38.6 months).

Table 3 summarizes the histologic features of OEMCa and ApEMCa. All
tumors showed a multinodular pattern of invasion typical of all EMCa
(Figure 1). Although the proportion of epithelial to myoepithelial
components varied both between and within tumors, all tumors were
decidedly biphasic even in areas of invasion. Papillary growth was noted
in 3 of 5 OEMCa (60%; Figure 2, A) but not in ApEMCa. Additionally,
luminal calcifications (Figure 2, B) were noted more frequently in OEMCa
(all cases) than in ApEMCa (only 1 of 5 cases; 20%). The prevalence of
perineural (OEMCa, 2 of 5, 40%; ApEMCa, 1 of 5, 20%) and angiolymphatic
(OEMCa, 1 of 5, 20%; ApEMCa, 2 of 5, 40%) invasion were, however,
similar, with the invasive areas retaining their biphasic nature,
although some tumor emboli were purely myoepithelial. No angioinvasive
or perineural invasive areas were purely ductal. For OEMCa, the mean
oncocytic component percentage was 73% (range, 60%-85%), whereas for
ApEMCa, the mean apocrine component percentage was 93% (range, 70%-95%).
In OEMCa, the epithelial component was nearly uniformly oncocytic, but
the myoepithelial component ranged from clear to oncocytic (Figure 3, A
and B). In ApEMCa, the myoepithelial component ranged from clear to pale
am phophilic, with 1 case showing oncocytic cytoplasm. Sebaceous
components (Figure 3, C) were only seen in OEM Ca (mean, 26%; range,
15%-40%). In ApEMCa, most tumor (3 of 5; 60%) showed a ductal component
with a tubular pattern (Figure 4, A), but 1 case showed solid overgrowth
(Figure 4, B), whereas another showed cribriform overgrowth (Figure 4,
C).

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The morphology of the ductal component is what was most useful
morphologically in the separation of OEMCa and ApEMCa. Key features
separating oncocytic epithelium and apocrine epithelium in these lesions
are shown in Figure 5. The lumina of OEMCa are smooth, whereas ApEMCa
typically demonstrates apical snouts. The cytoplasm of OEMCa is
uniformly granular and deeply eosinophilic, whereas ApEMCa ranges from
eosinophilic and granular to pale and vacuolated, often coarsely
vacuolated with intracytoplasmic lumina. An overall assessment of
nuclear atypia showed that both lesions were similar, however, there was
greater nuclear size variation in ApEMCa than in OEMCa. The luminal
cells in ApEMCa tended to have more vesicular nuclei with more prominent
nucleoli than OEMCa.

On ultrastructural examination, all 3 OEMCa examined showed
abundant mitochondria in the epithelial components and, to a lesser
degree, in the myoepithelial components. Organellar detail was difficult
to ascertain because the material was originally processed for paraffin
sections. Sebaceous cells were characterized by numerous vacuoles
indenting the nucleus and scattered mitochondria. The lipid droplets
were not evident because the examined material was derived from the
paraffin blocks (Figure 6). The 1 ApEMCa examined demonstrated a luminal
border with long irregular villi and scattered decapitation secretions
(Figure 7). The cytoplasm showed varying degrees of vacuolar change.
Mitochondria were noted as well, although not to the same extent as in
OEMCa.

Both OEMCa and ApEMCa expand the already wide differential
diagnosis that must be considered when approaching the pink tumors of
the head and neck, consisting of true oncocytic lesions, such as
oncocytoma and oncocytic carcinoma, and prominently oncocytoid tumors,
such as salivary duct carcinoma. (4,5) At first glance, the distinction
between these 2 subtypes of EMCa appears to be equivalent to splitting
hairs. But the morphologic and immunophenotypic differences on careful
examination are reproducible and cannot be ignored. Oncocytic-sebaceous
epithelial-myoepithelial carcinoma comprises true oncocytes, namely,
cells in which the granular eosinophilia is a result of the abundance of
mitochondria. Apocrine epithelial-myoepithelial carcinoma, on the other
hand, consists of cells that are oncocytoid; their eosinophilia is
likely a result of a combination of factors: protein content, secretory
vacuoles, with perhaps a minor contribution from mitochondria.
Additionally, the other morphologic features and immunopositivity for AR
and GCDFP-15 are compatible specifically with apocrine differentiation.
It is important not to mistake either of these EMCa variants for benign
salivary gland lesions, such as oncocytoma/oncocytosis, although OEMCa
is more likely to be confused with these entities. On the other hand, it
is important not to overinterpret ApEMCa as salivary duct carcinoma,
either de novo or ex pleomorphic adenoma, because the implied prognosis
and treatment will be dramatically altered. An algorithmic approach is
shown for distinguishing select entities in the differential diagnosis
in Figure 11.

[FIGURE 10 OMITTED]

The key step in the decision-making tree is the recognition of a
biphasic pattern. This is done both morphologically and
immunophenotypically. A caveat to using immunohistochemistry alone is
that oncocytomas/oncocytic carcinomas and salivary duct carcinoma in
situ will have an outer delimiting basal or myoepithelial layer
highlighted by immunostains. However, in oncocytoma/oncocytic carcinoma,
the transition from this outer layer is gradual and does not result in 2
reproducibly distinct components, (7) and in salivary duct carcinoma in
situ, this layer is indistinct and not considered part of the neoplastic
process. (8) For these reasons, these entities are not considered truly
biphasic.

The second step toward arriving at OEMCa and ApEMCa is the
confirmation that the outer layer in a biphasic tumor is indeed
myoepithelial. The outer myoepithelial cell layers are typically
positive for p63 and a muscle marker, such as smooth muscle actin,
calponin, or smooth muscle myosin heavy chain. S100 can be used, but we
do not favor using this marker based on our previous experience with its
infidelity as a myoepithelial marker. (4) This immunophenotype separates
OEMCa and ApEMCa from other bilayered oncocytic lesions, namely Warthin
tumor and oncocytic cystadenoma/cystadenocarcinomas, where the outer
cell layers are only typically p63 positive, that is, basal. (9,10) In
addition, the outer cell layers in Warthin tumors and oncocytic
cystadenoma/cystadenocarcinomas are morphologically less prominent than
those of the EMCa variants.

[FIGURE 11 OMITTED]

The final step in delineating OEMCa and ApEMCa is subtle and
challenging. Entities still in the differential are sclerosing
polycystic adenosis and pleomorphic adenoma (PA), with
oncocytic/apocrine components or even salivary duct carcinoma arising in
PA. Here, it is the morphology and arrangement of the outer
myoepithelial layer that separates these lesions. In all EMCa variants,
the outer myoepithelial cells have an activated appearance with cell and
nuclear size that is more similar and often even larger than those of
the ductal component. (3,4) This feature effectively separates these
EMCa variants from sclerosing polycystic adenosis, where the outer layer
is attenuated. (11) Key features in distinguishing OEMCa and ApEMCa from
PAs with oncocytic or apocrine change are the presence of invasion in
the EMCa variants, chondroid elements in PAs, and dispersion or
streaming of the myoepithelial components into a myxoid stroma in PA.
Another consideration with ApEMCa is the distinction from salivary duct
carcinoma arising in PA. In the former, the biphasic nature is retained,
even in the invasive areas, whereas in the latter, the salivary duct
carcinoma is the only invasive constituent. Additionally, although
showing atypia and even p53 reactivity in one case, the ductal component
of ApEMCa had a low proliferation index and no evidence of necrosis.

The initial, and even the most recent, World Health Organization
definition of EMCa requires that the myoepithelial component have clear
cells. (1-3) Many tumors with a nonclear myoepithelial component exist
that are, from a behavioral standpoint, no different from classic EMCa,
just as spindled, plasmacytoid, and epithelioid-patterned myoepithelial
carcinomas are no different from their clear-cell counterparts. (4,12)
Thus, taxonomically speaking, it is logical to simply include biphasic
invasive tumors without a clear-cell myoepithelial component as variants
of EMCa.

The question still remains as to the similarities and differences
in the tumorigenesis of all variants of EMCa. Because these are rare
variants of a rare tumor, the underlying molecular events cannot be
currently studied. Any proposed models of tumor development and
progression are admittedly speculative. Nonetheless, we will make an
attempt at this based on available morphologic and immunophenotypic
data.

One point is that the mean age for OEMCa is about a decade higher
than for EMCa overall, suggesting that, similar to other oncocytic
lesions, (13,14) senescence may play a role in the pathogenesis of this
variant. On the other hand ApEMCa is somewhat of a conundrum. The AR+,
GCDFP-15+, HER2/neu-positive phenotype essentially defines the salivary
duct carcinoma class of lesions. There is evidence to suggest that the
immunophenotype precedes the development of malignancy. Recently, a
small percentage of PA without overt salivary duct carcinoma, invasive
or in situ, were noted to show AR and HER-2/ neu positivity, (15) This
finding can be expected in PA because salivary duct carcinoma often
evolves from PA, (16) but this phenotype in the ductal component of EMCa
is rather unexpected.

However, salivary duct carcinomas arising from other tumors have
been documented, and an EMCa-salivary duct carcinoma hybrid tumor has
indeed been described. (17) In light of the paucity of mitoses, absence
of necrosis, and low Ki-67 proliferation index in our ApEMCa, we did not
label these tumors as EMCa-salivary duct carcinoma hybrid tumors.
Nevertheless, these findings suggest that apocrine features preceding
overt salivary duct carcinoma are possible in EMCa as well. Another
argument supporting the feasibility of this phenomenon, is the
observation that EMCa is the most common tumor partner in hybrid
salivary gland carcinomas. (17,18) Because the other partner is
typically higher grade, the implication is that the EMCa serves as the
progenitor or base tumor.

When comparing OEMCa and ApEMCa, the morphologic differences are
slight but reproducible, and the differential immunohistochemical
profile and even ultrastructural features are quite useful in the
distinction. Of interest, sebaceous elements were commonly seen in OEMCa
but not ApEMCa. This is surprising when comparing these lesions to skin
adnexal tumors, where apocrine lesions commonly display sebaceous
elements. (19) It is difficult to draw conclusions based on such a low
number of cases; we simply may not yet have encountered sebaceous
differentiation in apocrine salivary lesions. However, one observation
suggesting that this difference may have a biologic basis is that in the
skin, both sebaceous and apocrine elements are likely driven
androgenically because they both are AR positive. But as we noted
previously, sebaceous elements in EMCa are negative4 for AR suggesting a
different evolution. With this being the case, the coexistence of
sebaceous elements with oncocytic rather than apocrine lesions can be
considered a reflection of the different set of rules governing
differentiation in salivary gland lesions.

In summary, we compare and contrast 2 relatively new variants of
EMCa--OEMCa and ApEMCa. These lesions add to the already sizeable
differential diagnostic litany for oncocytic and oncocytoid lesions of
the head and neck. Based on limited follow-up, these variants appear to
have the same multinodular growth pattern and indolent behavior as their
classic EMCa counterparts. The mere existence of these morphologic
variants raises several questions regarding tumorigenesis in EMCa, but
their molecular pathogenesis will likely remain elusive for some time.